Bluetongue virus (BTV) is a multi-layered, double-stranded, arthropod borne RNA virus and is the type member of the Orbiviruses, within the family Reoviridae. As such it shares a virus family relationship with several other scientifically and medically important viruses such as Reoviruses and Rotaviruses. As a result of NIH funded projects to date BTV has been studied extensively as an agent of economically important disease and also, increasingly, as a model system for viruses of the entire family. Formidable progress has been achieved in sequence, biochemical and most recently, structural fields such that, today, BTV is one of the most well understood of all viruses. Certain areas of BTV biology remain unclear however, in particular, the question of how virus and host protein together orchestrate a successful infectious cycle. This proposal is to maintain and extend the leading position of BTV towards our long term goal of understanding, in totality, the infection course of, and the host involvement with, a non-enveloped arthropod-borne virus. As a result of its model status the achievement of this goal for BTV will benefit a range of related viruses. The proposal focuses on four BTV proteins whose sequential role in viral processes from the entry of the virus to its exit from the cell remain undefined. The virion outer capsid protein, VP5, which appears to have fusigenic activity and facilitate virus entry into the cytoplasm is key to understanding the initiation of infection. The non-structural protein NS 1, that forms tubules (unique to orbiviruses) and is believed to recruit virion components into replication centres, is central to a productive virus life cycle. The phosphoprotein NS2, which has RNA binding ability is crucially important in the generation of appropriately formed progeny virions, and the NS3, together with a cellular protein, is a factor in efficient virus release. The functions of these proteins are fundamental to the success of the BTV life cycle yet precisely how each protein completes its role and to what extent host factors are involved remain largely unknown. The proposal builds on the success of our previous NIH award and will make use the extensive range of BTV and related reagents that have been enabled by our fundings. These include all BTV gene cloned, sequenced and verified by protein expression; sources of highly purified biologically active BTV proteins; a range of high titre monospecific antisera; monoclonal antibodies; sequence specific nucleic acid probes; and three dimensional structural information. Together with the proposed experimental programme, our reagent and knowledge base will allow us to complete the tasks we have specified to offer new insights into virus replication and new suggestions for effective control.